Vitamin k for prevention and treatment of skin rash secondary to anti-egfr therapy

ABSTRACT

The invention provides methods and compositions for treating and preventing a skin rash secondary to anti-epidermal growth factor receptor (EGFR) therapy, where the method comprises applying a vitamin K analog or a phosphatase inhibitor to the skin.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 12/788,015filed on May 26, 2010 which is a divisional of U.S. application Ser. No.11/886,803, filed on Jan. 9, 2009, which is a National Stage entry ofPCT/US2006/014158, filed on Apr. 12, 2006, which claims the benefit ofU.S. Provisional Application No. 60/671,563, filed on Apr. 15, 2005, theentire contents of each of these applications is hereby incorporatedherein by reference.

STATEMENT OF GOVERNMENT SUPPORT

The invention disclosed herein was made with U.S. Government supportunder National Institutes of Health grant numbers CA 84119 and 91784.Accordingly, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparenthesis. Full citations for these references may be found at the endof the specification immediately preceding the claims. The disclosuresof these publications are hereby incorporated by reference in theirentireties into the subject application to more fully describe the artto which the subject application pertains.

The epidermal growth factor receptor (EGFR) is a transmembrane proteinexpressed in all epithelial surfaces. It plays an importantphysiological role in epithelial repair and regeneration. Epidermalgrowth factor (EGF) is a peptide secreted by salivary glands and otherglands associated with epithelial surfaces that binds to a specific areain the extracellular domain of EGFR. Upon binding it generates a signalthat is transmitted inside the cell. The first intracellular event as aresult of EGF binding is a conformational change of the intracellulardomain of EGFR that allows adenosine 5′-triphosphate (ATP) to enter theso-called tyrosine kinase (TK) domain, a pocket that contains a tyrosineresidue, and donate a phosphate group to the tyrosine residue. Theintracellular EGFR carrying a phosphorylated tyrosine becomes capable ofassociating with other intracellular proteins and originates a series ofbiochemical reactions that propagate downstream through a very complexnetwork. The best known arms of this network are the mitogen-activatedprotein kinase (MAPK) pathway, which results in tumor cell division uponactivation, and the AKT pathway, which results in enhanced cell survivalupon activation. The results of EGFR activation are therefore increasedcell proliferation and enhanced cellular tolerance to different insults.

Many tumors overexpress EGFR compared to vicinal normal tissues or theepithelial surface from which they originate or have a mutated versionof EGFR, intrinsically activated or with an enhanced susceptibility toactivation. Such overexpression is thought to be one of the manymechanisms by which tumor cells gain a growth advantage, a keycharacteristic of the malignant phenotype. Consequently, blocking theEGFR signaling pathway is thought to be a rational strategy for thetreatment of many human malignancies. There are basically two ways toinhibit upstream the EGFR signaling pathway: 1) preventing EGF and othernatural peptide ligands from binding to the extracellular EGFR domain bythe use of specific monoclonal antibodies, and 2) preventing ATP andother phosphate donors from entering the TK pocket of the intracellularEGFR domain by the use of small molecules that structurally fit verywell into the pocket (the so-called EGFR TK inhibitors).

After years of discovery efforts and clinical evaluation, EGFRinhibitors have been recently shown to be active antitumor agentsagainst a variety of solid tumors including but not limited tocolorectal carcinoma, non-small cell lung cancer, head and neck cancerand malignant gliomas (Conen et al., 2003; Lage et al., 2003; Lorusso,2003; Vanhoefer et al., 2004). Clinical benefit defined as relief ofsymptoms or prolongation of survival has been so far demonstrated withthe anti-EGFR antibody cetuximab (Erbitux®) and the EGFR tyrosine kinase(TK) inhibitors gefitinib (Iressa®) and erlotinib (Tarceva®). Manyadditional agents belonging to this class are being developed. As oftoday, FDA approved indications include chemorefractory colorectalcarcinoma and non-small cell lung cancer, head and neck carcinoma, andpancreatic carcinoma. Many clinical studies using these agents alone orin combination, for refractory or chemo naive patients with a variety ofother malignant diseases are in progress. It is anticipated that as manyas 200,000 patients in the USA may receive these agents every year inthe near future.

Although these agents do not cause life threatening toxicities, themajor side effect is a skin rash that occurs in 60-70% of patients,affects mostly face and trunk, and causes discomfort and unfavorablecosmetic changes in many cases. Main symptoms caused by the skin rashare itching, dryness, and secondary infection. The occurrence andintensity of the rash are clearly dose-related and the median time ofoccurrence is 10 days after initiation of therapy. About 10% of patientsdiscontinue therapy due to skin toxicity.

Most clinical studies have shown that most patients who develop skinrash as a result of anti-EGFR therapy tend to live longer. It is thoughtthat the occurrence of skin rash requires both effective EGFR inhibitionin the skin and a competent immune system that is able to respond to thetissular insult caused by such inhibition with an appropriateinflammatory response. As there is growing evidence that the skin rashis a surrogate indicator of antitumor efficacy and clinical benefit(Cohen et al., 2003), increasing dosing of anti-EGFR agents to cause askin rash may become a common practice. If that is the case, treatmentof the skin rash will also become of increasing importance. As a result,treatment of the skin rash is also becoming of increasing importance.There are no rational, scientifically proven and clinically effectivemethods available for the treatment of this form of skin rash. Topicalor systemic antibiotics, anti-inflammatory agents, retinoids, topicallubricants, and other types of remedies have been tried in an empiricalfashion with poor or inconsistent results. As of today there is noestablished therapy or prevention for this new skin disease, a conditionthat is expected to affect about 150,000 individuals in the USA everyyear in the very near future.

Vitamin K has been used for treatment of blood vessel disorders of theskin, cosmetic skin treatment, and skin treatment following lasertreatment (U.S. Pat. No. 5,510,391; U.S. Patent Application PublicationNo. US 2003/0170187; PCT International Publication Nos. WO 97/39746, WO02/13780, WO 03/101415, WO 2004/019923; Lou et al., 1999; Shah et al.,2002). However, topical administration of vitamin K3 can causedermatitis (Page et al., 1942) and vesiculation of the skin (Ulbrich,1961).

SUMMARY OF THE INVENTION

The present invention provides a method for treating a skin rashsecondary to an anti-epidermal growth factor receptor (EGFR) therapy ina subject receiving said therapy, the method comprising applying avitamin K analog to the skin rash in an amount effective to treat theskin rash. The invention also provides a method for preventing a skinrash secondary to an anti-epidermal growth factor receptor (EGFR)therapy in a subject receiving said therapy, the method comprisingapplying a vitamin K analog to the skin in an amount effective toprevent the skin rash.

The invention provides a pharmaceutical composition comprising an amountof a vitamin K analog effective to treat or prevent a skin rashsecondary to an anti-epidermal growth factor receptor (EGFR) therapy.

The invention also provides a method for identifying a compound that maybe useful for treating and/or preventing a skin rash secondary to ananti-epidermal growth factor receptor (EGFR) therapy, the methodcomprising determining whether the compound activates any one of EGFR,Akt, and Signal Transducer and Activator of Transcription-3 (STAT-3),wherein activation of EGFR, Akt or Stat-3 by the compound is indicativethat the compound is effective for treating and/or preventing the skinrash.

The present invention is also directed to a method for treating a skinrash secondary to an anti-epidermal growth factor receptor (EGFR)therapy in a subject receiving said therapy, the method comprisingapplying a phosphatase inhibitor to the skin rash in an amount effectiveto treat the skin rash. The invention further provides a method forpreventing a skin rash secondary to an anti-epidermal growth factorreceptor (EGFR) therapy in a subject receiving said therapy, the methodcomprising applying a phosphatase inhibitor to the skin in an amounteffective to prevent the skin rash.

The invention also provides a pharmaceutical composition comprising anamount of a phosphatase inhibitor effective to treat or prevent a skinrash secondary to an anti-epidermal growth factor receptor (EGFR)therapy.

The invention also provides a method for treating a skin or mucosalulceration in a subject, the method comprising applying to the skin ormucosal ulceration an amount of a vitamin K analog effective to treatthe skin or mucosal ulceration, wherein the vitamin K analog activatesEGFR. The invention further provides a method for treating a skin ormucosal ulceration in a subject, the method comprising applying to theskin or mucosal ulceration an amount of a phosphatase inhibitoreffective to treat the skin or mucosal ulceration, wherein thephosphatase inhibitor inhibits dephosphorylation of EGFR.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1C. Effects of Vitamins K1, K2 and K3 (VK1, VK2, VK3) on thephosphorylation of EGFR (A), AKT (B) and Stat-3 (C) in human A431 vulvarcarcinoma cells. A431 cells (1×10⁶ cells/well) were plated in a 6-wellplate, and incubated in RPMI-1640 medium with 10% serum overnight. Cellswere exposed to the indicated concentrations of VK1, VK2 and VK3 for 2hours. Following exposure, cells were washed twice with cold phosphatebuffered saline (PBS) solution, and lysed with 50 μl of lysis buffer.Equal amounts of lysate (30 μg of protein) were subjected to a 7.5% or a12% SDS-PAGE. EGFR, phos-EGFR, AKT, phos-AKT, Stat-3, and phos-Stat-3were detected by immunoblotting analysis using corresponding antibodies.Vitamin K3 stimulated EGFR, Akt and Stat-3. Vitamin K1 also activatedEGFR, whereas Vitamin K2 had little or no effect.

FIG. 2. Vitamin K3 (menadione) prevents the inhibition of EGFRphosphorylation caused by erlotinib, an EGFR tyrosine kinase inhibitor,in A431 cells. A431 cells (1×10⁶ cells/well) were plated in a 6-wellplate and incubated in RPMI-1640 medium without fetal bovine serum for24 hours. After starving, cells were exposed to different concentrationsof vitamin K3 alone (0.01-0.5 mM) or with 2 μM erlotinib for 2 hours, orstimulated with 100 ng/ml of EGF for 5 min. After exposure, cells werewashed twice with cold PBS solution, and lysed with 50 μl of lysisbuffer. Equal amounts of lyaste (30 μg of protein) were subjected to a7.5% SDS-PAGE. EGFR and phosphorylated EGFR were detected byimmunoblotting analysis using corresponding antibodies.

FIG. 3. Effect of VK3 (menadione) on erlotinib-induced cell growthinhibition in A431 cells. Cells were plated in a 96-well plateovernight. Cells were exposed to different concentrations of erlotinibalone or in combination with 50 μM or 100 μM VK3 (menadione) for 72hours. After exposure, cells were harvested by trypsinization, and thecell viability was assessed by cell counting using a hemacytometer. Eachpoint represents the mean of two independent experiments.

FIG. 4A-4B. VK3-induced phosphorylation of EGFR is reversible in A431cells. Cells were exposed to 0.1 mM VK3 for 1 hour. Following exposure,cells were washed three times with cold medium and then reincubated inVK3-free fresh medium with 10% fetal bovine serum for the indicatedtimes. Cells were harvested and lysed with lysis buffer. Equal amountsof cell lysate were subjected on 7.5% SDS-PAGE (A). The total EGFR andphosphorylated EGFR (p-EGFR) were detected by immunoblot analysis usingcorresponding antibodies. Control (Con) means cells were not exposed toVK3. The relative p-EGFR was expressed as the intensity of p-EGFR bandsin each time points measured by a laser densitometry compared with thatin control as one (B).

FIG. 5. Vitamin K3 inhibits phosphatase activity. A431 cells wereexposed to 0.1 mM VK3 for 1 hour. After exposure, cells were washedthree times with cold medium and then reincubated in VK3-free mediumwith 10% fetal bovine serum for the indicated time. Cells were harvestedand cell extracts were prepared for the assay of phosphatase activityusing pNPP (4-nitrophenyl phosphate disodium) as a substrate. Control(Con) means the phosphatase activity in cells without exposure to VK3.The relative phosphatase activity was expressed as compared with that incontrol as one. VK3-induced phosphatase inhibition is reversiblefollowing removal of VK3.

FIG. 6. Effect of Vitamin K3 (menadione) on erlotinib-induced inhibitionof EGFR phosphorylation in mouse skin. Vitamin K3 rescueserlotinib-induced inhibition of EGFR phosphorylation in mouse skintissue. Mice were administrated P.O. daily 50 mg/kg of erlotinib; mouseskin was smeared with VK3 15 mM ethanol solution. After treatment, themice were sacrificed and skin tissue was taken. 100 mg skin tissue wasused for the preparation of tissue extracts. Equal amounts of skintissue extracts (80 μg of protein) were subjected on a 7.5% SDS-PAGE.Total EGFR and p-EGFR were detected by immunoblot analysis.

FIG. 7. Effect of VK3, Cream B and Cream VK on EGFR phosphorylation inmouse skin tissue. The mice were smeared with 30 mM or 100 mM VK3 (inethanol), or with the same doses of Cream B or Cream VK. Followingtreatment, mice were sacrificed and skin tissues were taken for thepreparation of tissue extracts. Equal amounts of tissue extracts (80 μgof protein) were subjected on a 7.5% of SDS-PAGE. Total EGFR and p-EGFRwere detected by immunoblot analysis using polyclonal anti-EGFR andp-EGFR antibodies. Cream B is an over the counter product (Baby Cakes)containing Vitamin K3 according to its label; the concentration is notspecified. Cream VK is a 5% Vitamin K cream available over the counter.In contrast to 30 mM or 100 mM VK3, the commercial creams do not show aneffect.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for treating a skin rashsecondary to an anti-epidermal growth factor receptor (EGFR) therapy ina subject receiving said therapy, the method comprising applying avitamin K analog to the skin rash in an amount effective to treat theskin rash.

As used herein, the term “treat” a skin rash secondary to anti-EGFRtherapy means to reduce or eliminate the skin rash, and/or to decreaseor eliminate the increase or spread of the skin rash on the subject'sskin.

Subjects receiving anti-EGFR therapy include patients receivingtreatment with an anti-EGFR antibody such as cetuximab and/or with anEGFR tyrosine kinase inhibitor such as gefitinib or erlotinib.

As used herein, a “vitamin K analog” is a compound selected from thegroup consisting of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone),vitamin K2 (2-methyl-3-hexaprenyl-1,4-naphthoquinone; menaquinone),vitamin K3 (2-methyl-1,4-naphthoquinone; menadione), vitamin K4(1,4-diacetoxy-2-methylnaphthalene), vitamin K5(4-amino-2-methyl-1-naphthalenol), vitamin K6, vitamin K7(3-methyl-4-amino-1-naphthol hydrochloride), menadione diphosphate,menadione sodium bisulfite, and compounds having the structure:

where R═H or a saturated or unsaturated aliphatic hydrocarbon; and R₁and R₂═H or CH₃.

Vitamin K3 and vitamin K1 are preferred vitamin K analogs. Vitamin K3 ismost preferred. Vitamin K3 has the structure:

Vitamin K1 has the structure:

Preferred forms of vitamin K1 are the trans isomer of vitamin K1 or anadmixture of the trans and cis isomers, since the cis isomer has littleor no biological activity (Matschiner et al., 1972).

Vitamins K1 and K2 occur naturally. Vitamins K3, K4, K5, K6, and K7 aresynthetic analogs. Processes have been described for preparing vitamin Kanalogs, e.g., U.S. Pat. Nos. 4,374,775, 4,906,411, 5,412,124,5,637,741, 5,770,774, and 6,579,994, and Sah (1949-50). Vitamins K1, K2and K3 are available, e.g., from Sigma-Aldrich.

The vitamin K analog can activate EGFR, Akt and/or Stat-3. Vitamin Kanalogs can be screened to identify those that activate EGFR, Akt and/orStat-3 using procedures described in the present application. As usedherein, to “activate” EGFR, Akt and/or Stat-3 means to increase thephosphorylated form of EGFR, Akt and/or Stat-3.

As used herein to treat a skin rash, the vitamin K analog is topicallyapplied to the skin. Vitamin K compounds are easily soluble in ethylalcohol and organic solvents and can be prepared in formulations fortopical use at desired concentrations. The vitamin K analog may beapplied to the skin using, e.g., creams, gels, emulsions, lotions,liquids and/or liposomes. The formulation can be a formulation thatprovides sustained-release of the vitamin K analog. Topical skinformulations can include, for example, ingredients which include but arenot limited to any of the following: ethyl alcohol, isopropyl alcohol,benzyl alcohol, cetyl alcohol, stearyl alcohol, lecithin granules,isopropyl palmitate, isopropyl myristinate, propyl paraben, methylparaben, a surfactant (e.g., Pluronic F-127 NF), Dowicil 200, mineraloil, natural oil, silicone oil, paraffin, glycerine, stearic aciddiester, glycerol stearate, stablizers such as cyclodextrin, andultraviolet (UV) filters.

Vitamin K analogs can be applied to the skin up to the maximum tolerateddose, which may vary from subject to subject and depend upon thespecific vitamin K analog used in treatment. The maximum tolerated dosecan be readily determined by one skilled in the art. Preferably, thevitamin K analog is applied to the skin at a concentration of at least100 μM or at least 15 μg/ml. Preferably, vitamin K3 is applied at aconcentration of 50 μg/ml to 200 μg/ml, more preferably 75 μg/ml to 100μg/ml. Preferably, vitamin K1 is applied to the skin at a concentrationof at least 450 μg/ml.

Treatment with the vitamin K analog can start, for example, on the dayof initiation of therapy with an anti-EGFR agent or as soon as a skinrash appears or as otherwise prescribed by a physician. The vitamin Kanalog is administered periodically during the course of therapy withthe anti-EGFR agent. The frequency of administration will vary accordingto the formulation, for example twice a day, daily or every other day oras otherwise prescribed by a physician. A sustained release formulationwill require less frequent administration than a formulation that doesnot provide sustained or slow release of the vitamin K analog.

The invention further provides a method for preventing a skin rashsecondary to an anti-epidermal growth factor receptor (EGFR) therapy ina subject receiving said therapy, the method comprising applying avitamin K analog to the skin in an amount effective to prevent the skinrash, as described herein for treatment of a skin rash. Skin rashsecondary to anti-EGFR therapy commonly occurs on the face and trunk.Preferably, the vitamin K analog is applied to areas of the body wherethe skin rash typically appears secondary to anti-EGFR therapy, such asthe face and trunk, or to an area of the body where the subject hasexperienced a skin rash during prior anti-EGFR therapy. In order toprevent the skin rash, the vitamin K analog can be applied to the skinstarting, for example, on the day of initiation of therapy with theanti-EGFR agent or as otherwise prescribed by a physician.

The invention also provides a pharmaceutical composition comprising anamount of any of the vitamin K analogs described herein effective totreat and/or prevent a skin rash secondary to an anti-epidermal growthfactor receptor (EGFR) therapy. The compositions can also include apharmaceutically acceptable carrier. As used herein, the term “carrier”encompasses standard pharmaceutical carriers such as ethyl alcohol andorganic solvents. In a preferred embodiment, the pharmaceuticalcomposition is formulated for topical administration. The vitamin Kanalog can be formulated in a sustained release formulation.

The invention also provides methods for identifying a compound that maybe useful for treating and/or preventing a skin rash secondary to ananti-epidermal growth factor receptor (EGFR) therapy, where the methodcomprises determining whether the compound activates any one or more ofEGFR, Akt, and Signal Transducer and Activator of Transcription-3(STAT-3), wherein activation of EGFR, Akt or Stat-3 by the compound isindicative that the compound is effective for treating and/or preventingthe skin rash. Activation of EGFR, Akt or Stat-3 can be determined,e.g., by using an antibody that is specific for the phosphorylated(activated) form of EGFR, Akt or Stat-3. The compound can be, forexample, a vitamin K analog. The compound can inhibit dephosphorylationof EGFR, Akt or Stat-3. The compound can be a phosphatase inhibitor.

The present invention is also directed to a method for treating a skinrash secondary to an anti-epidermal growth factor receptor (EGFR)therapy in a subject receiving said therapy, the method comprisingapplying a phosphatase inhibitor to the skin rash in an amount effectiveto treat the skin rash. The invention further provides a method forpreventing a skin rash secondary to an anti-epidermal growth factorreceptor (EGFR) therapy in a subject receiving said therapy, the methodcomprising applying a phosphatase inhibitor to the skin in an amounteffective to prevent the skin rash. Preferably, the phosphataseinhibitor inhibits dephosphorylation of EGFR. A variety of phosphataseinhibitors have been described (for example, Bae et al. 2004, Gerling etal. 2004, Lee and Burke 2003, Liem et al. 2004, U.S. Pat. Nos. 5,155,031and 6,428,949 B1, U.S. Patent Application Publication No. US2004/0138218 A1). The phosphatase inhibitor may be applied to the skinusing, e.g., creams, gels, emulsions, lotions, liquids and/or liposomes.The formulation can be a formulation that provides sustained-release ofthe phosphatase inhibitor. The phosphatase inhibitor can be applied tothe skin up to the maximum tolerated dose, which may vary from subjectto subject and depend upon the specific phosphatase inhibitor used intreatment. The maximum tolerated dose can be readily determined by oneskilled in the art.

The invention also provides a pharmaceutical composition comprising anamount of a phosphatase inhibitor effective to treat or prevent a skinrash secondary to an anti-epidermal growth factor receptor (EGFR)therapy. Preferably, the phosphatase inhibitor inhibitsdephosphorylation of EGFR. Preferably, the pharmaceutical composition isformulated for topical administration. The phosphatase inhibitor can beformulated in a sustained release formulation.

The invention also provides a method for treating a skin or mucosalulceration in a subject, the method comprising applying to the skin ormucosal ulceration an amount of a vitamin K analog effective to treatthe skin or mucosal ulceration, wherein the vitamin K analog activatesEGFR. The invention further provides a method for treating a skin ormucosal ulceration in a subject, the method comprising applying to theskin or mucosal ulceration an amount of a phosphatase inhibitoreffective to treat the skin or mucosal ulceration, wherein thephosphatase inhibitor inhibits dephosphorylation of EGFR. Skin andmucosal ulcerative conditions include, for example, ulcerative colitisand oral mucosistis, which can be induced by chemotherapy and/orradiotherapy. For ulcerative colitis, the vitamin K analog or thephosphatase inhibitor can be administered for example by enemaadministration. Local therapies by enema with steroids and otherantiinflammatory agents are currently standard of care for ulcerativecolitis. For oral mucosistis, e.g., an adhesive gel or mouth washcontaining the vitamin K analog or the phosphatase inhibitor can beused.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods

Two human cell lines that overexpress non-mutated EGFR were used: A431vulvar carcinoma and HN5 head and neck carcinoma. Cells were exposed todifferent concentrations of different vitamin K analogs in the presenceof one of three EGFR inhibitors: the anti-EGFR monoclonal antibodycetuximab (Erbitux®) and the EGFR tyrosine kinase inhibitors (TKIs)erlotinib (Tarceva®) and gefitinib (Iressa®). Cytotoxicity was assessedby cell counting. Cells were lysed and the effects of different vitaminK analogs on activation of EGFR, the kinase Akt, and Signal Transducerand Activator of Transcription-3 (STAT-3) were assessed by western blotanalysis using antibodies for the phosphorylated (activated) forms ofEGFR, Akt and Stat-3. Polyclonal antibodies of anti-EGFR, anti-phos-EGFR(Tyr1068), anti-AKT, anti-phos-AKT (Ser473), anti-Stat-3, andanti-phos-Stat-3 (Tyr-705) were purchased from Cell Signaling (Beverly,Mass.). Vitamins K1, K2, and K3 were purchased from Sigma-Aldrich (StLouis, Mo.). Activation of EGFR, Akt and Stat-3 were assessed in thepresence and absence of anti-EGFR treatments of the cells. Activation ofEGFR by vitamin K analog was also assessed using mouse skin, which washomogenized and lysed after treatment with the vitamin K analog.Phosphatase activity was determined using pNPP (4-nitrophenyl phosphatedisodium) as a substrate.

Results

The effects of Vitamins K1, K2 and K3 on activating (phosphorylating)EGFR, Akt and Stat-3 obtained from lysed A431 cells are shown in FIG.1A-1C. Vitamin K3 activated EGFR and downstream intracellular signalingcomponents Akt and Stat-3. Effects were clearly seen at a Vitamin K3concentration of 100 μM, or about 17 μg/ml. Vitamin K1 was onlyeffective for activating EGFR and Akt at the highest concentration used(1000 μM or about 450 μg/ml). Vitamin K2 had little or no effect at theconcentrations used (up to 1000 μM). Thus, Vitamin K3 is a much morepotent stimulant of EGFR, Art and Stat-3 than Vitamin K1 or Vitamin K2.Application of Vitamin K3 to normal mouse skin also activated EGFR.

Vitamin K3 activation of EGFR, Stat-3 and Akt was tested in the presenceof anti-EGFR treatments. A431 cells or HN5 cells stored in serum freemedium were treated with the EGFR tyrosine kinase inhibitor erlotinib (2μM) and with Vitamin K3 (0.01-0.5 mM). Vitamin K3 activated EGFR (FIG.2) and Stat-3 (at concentrations of 0.1 and 0.5 mM vitamin K3), and Akt(at a concentration of 0.5 mM vitamin K3) in the presence of erlotinib.Similarly, Vitamin K3 (0.1 and 0.5 mM) also activated EGFR, Stat-3 andAkt in the presence of the anti-EGFR monoclonal antibody cetuximab.Vitamin K3 also rescues erlotinib-induced inhibition of EGFRphosphorylation in mouse skin tissue, as shown in FIG. 6.

Cells were treated with excess EGF to determine if EGF could preventEGFR inhibition by different anti-EGFR treatments. EGF was effective inpreventing EGFR inhibition by the anti-EGFR monoclonal antibodycetuximab, which acts at the extracellular portion of the EGFR. Incontrast, treatment of cells with EGF (2 μg/ml) does not prevent EGFRinhibition by the EGFR tyrosine kinase inhibitor erlotinib, which actsat the intracellular portion of the EGFR (FIG. 2).

Exposure of human A431 vulvar carcinoma cells to the EGFR tyrosinekinase inhibitor erlotinib results in cell death. Co-incubation of thecells with Vitamin K3 (50-100 μM) decreased the cytotoxicity of the EGFRinhibitor by 4-6 fold. FIG. 3 illustrates results obtained with twodifferent concentrations of Vitamin K3 in rescuing cells treated witherlotinib. The effects of vitamin K3 are reversible. FIG. 4A-4B showsthat vitamin K3 induced phosphorylation of EGFR is reversible in A431cells.

Vitamin K3 acts at least in part by inhibiting phosphatase activity, asshown in FIG. 5. The effect is reversible 2-3 hours after removal ofVitamin K3.

The effect of Vitamin K3 was examined in the skin of animals receivingthe EGFR TK inhibitor erlotinib 100 mg/kg×10 days (days 1-10). Comparedto control skin in mice not receiving erlotinib, the skin treated withVitamin K3 in animals receiving erlotinib showed significant p-EGFRexpression, whereas the skin not treated with Vitamin K3 in the sameanimals showed downregulation of p-EGFR. (FIG. 6). The skin of mice wassmeared with Vitamin K3 in ethanol (15 mM BID×5 days on days 5 to 10).The effect of Vitamin K3 was compared to that of two over-the-counterskin creams. Cream B (Baby Cakes) is an over-the-counter productcontaining Vitamin K3 according to its label; the concentration is notspecified. Cream VK is a 5% Vitamin K cream available over-the-counter.The effect of VK3, Cream B and Cream VK on EGFR phosphorylation in mouseskin tissue is shown in FIG. 7. The mice were smeared with Vitamin K3(in ethanol) 30 mM or 100 mM×3 doses every 12 hours, or with the samedoses of Cream B or Cream VK. In contrast to 30 mM or 100 mM VK3, whichactivates EGFR, the commercial creams do not show an effect.

Discussion

The present application discloses that Vitamin K3 can prevent the EGFRinhibitory effects of anti-EGFR therapy in tumor cell lines ofepithelial origin that overexpress EGFR. The EGFR axis in these cells isrepresentative of the EGFR axis in skin keratinocytes. Vitamin K3 had astimulatory effect on the EGFR intracellular signaling pathway, whichwas effective even in the presence of anti-EGFR therapy. Vitamin K3 alsoactivated EGFR from normal mammalian (mouse) skin. Weaker stimulatoryeffects on EGFR were observed with Vitamin K1 but not with Vitamin K2.The present results demonstrate the unexpected effectiveness of VitaminK3, in comparison to Vitamin K1 or K2 or to two over-the-counter VitaminK skin creams, in combating anti-EGFR therapy. The use of vitamin K3 fortreatment and prevention of a skin rash as disclosed in the presentapplication is counterintuitive in view of previous reports that topicaladministration of vitamin K3 causes dermatitis (Page et al., 1942) andvesiculation of the skin (Ulbrich, 1961). Vitamin K3 was also shown toinhibit cellular phosphatase activity, thus providing for the use ofphosphatase inhibitors for treatment and prevention of skin rash andrelated skin or mucosal disorders.

REFERENCES

-   Bae E Y, Oh H, Oh W K, Kim M S, Kim B S, Kim B Y, Sohn C B, Osada H,    Ahn J S. A new VHR dual-specificity protein tyrosine phosphatase    inhibitor from Dendrobium moniliforme. Planta Med. 70(9):869-70,    2004.-   Cohen E E, Rosen F, Stadler W M, Recant W, Stenson K, Huo D, Vokes    E E. Phase II trial of ZD1839 in recurrent or metastatic squamous    cell carcinoma of the head and neck. J Clin Oncol. 21(10):1980-7,    2003.-   Gerling N, Culmsee C, Klumpp S, Krieglstein J. The tyrosine    phosphatase inhibitor orthovanadate mimics NGF-induced    neuroprotective signaling in rat hippocampal neurons. Neurochem Int.    2004 June; 44(7):505-20, 2004.-   Lage A, Crombet T, Gonzalez G. Targeting epidermal growth factor    receptor signaling: early results and future trends in oncology. Ann    Med. 35(5):327-36, 2003.-   Lee K, Burke T R Jr. CD45 protein-tyrosine phosphatase inhibitor    development. Curr Top Med. Chem. 3(7):797-807, 2003.-   Liem D A, Gho C C, Gho B C, Kazim S, Manintveld O C, Verdouw P D,    Duncker D J. The tyrosine phosphatase inhibitor    bis(maltolato)oxovanadium attenuates myocardial reperfusion injury    by opening ATP-sensitive potassium channels. J Pharmacol Exp Ther.    2004 June; 309(3):1256-62. Epub 2004 Mar. 1.-   LoRusso P M. Phase I studies of ZD1839 in patients with common solid    tumors. Semin Oncol. 30 (1 Suppl 1):21-9, 2003.-   Lou W W, Quintana A T, Geronemus R G, Grossman M C. Effects of    topical vitamin K and retinol on laser-induced purpura on    nonlesional skin. Dermatol Surg. 25(12):942-4, 1999.-   Matschiner J T, Bell R G. Metabolism and vitamin K activity of cis    phylloquinone in rats. J. Nutr. 102(5):625-629, 1972.-   Page, R C and Bercovitz, Z. Dermatitis from topical administration    of 2-methyl-1:4-naphthoquinone (synthetic vitamin K analogue).    Am. J. Med. Sci. 203: 566-569, 1942.-   Sah, P P. Synthesis of 3-methyl-4-amino-1-naphthol hydrochloride    (vitamin K7) and related vitamin-K-active compounds. Z Vitam Horm    Fermentforsch 3(3-4):324-45, 1949-1950.-   Shah N S, Lazarus M C, Bugdodel R, Hsia S L, He J, Duncan R,    Baumann L. The effects of topical vitamin K on bruising after laser    treatment. J Am Acad Dermatol. 47(2):241-4, 2002.-   Ulbrich, A P. Topical application of menadione, a synthetic vitamin    K: preliminary report. J. Am. Osteopathic Assoc. 60: 370-374, 1961.-   Vanhoefer U, Tewes M, Rojo F, Dirsch O, Schleucher N, Rosen O,    Tillner J, Kovar A, Braun A H, Trarbach T, Seeber S, Harstrick A,    Baselga J. Phase I study of the humanized antiepidermal growth    factor receptor monoclonal antibody EMD72000 in patients with    advanced solid tumors that express the epidermal growth factor    receptor. J Clin Oncol. 22(1):175-84, 2004.-   PCT International Publication No. WO 97/39746, Oct. 30, 1997, Method    of and composition for treating disorders of the skin using vitamin    K.-   PCT International Publication No. WO 02/13780, Feb. 21, 2002,    Cosmetic cream.-   PCT International Publication No. WO 03/101415, Dec. 11, 2003,    Compositing comprising vitamin K.-   PCT International Publication No. WO 2004/019923, Mar. 11, 2004,    Compositions comprising vitamin K for treating or preventing    age-related stiffening of arteries.-   U.S. Pat. No. 4,374,775, issued Feb. 22, 1983, Dotz K H, Process for    preparing vitamin K.-   U.S. Pat. No. 4,906,411, issued Mar. 6, 1990, Shinnaka A et al.,    Process for producing 2-methyl-1,4-naphthoquinone.-   U.S. Pat. No. 5,155,031, issued Oct. 13, 1992, Posner et al., Use of    pervanadate as an inhibitor of phosphotyrosine phosphatase.-   U.S. Pat. No. 5,412,125, issued May 2, 1995, Hamamura K et al.,    Preparation process of naphthoquinone derivative and intermediate    for the preparation thereof.-   U.S. Pat. No. 5,510,391, issued Apr. 23, 1996, Elson M L, Method of    treating blood vessel disorders of the skin using vitamin K.-   U.S. Pat. No. 5,637,741, issued Jun. 10, 1997, Matsumoto Y et al.,    Process for producing 2-methyl-1,4-naphthoquinone.-   U.S. Pat. No. 5,770,774, issued Jun. 23, 1998, Joo Y J et al.,    Method for preparing 2-methyl-1,4-naphthoquinone (vitamin K₃).-   U.S. Pat. No. 6,428,949 B1, issued Aug. 6, 2002, Bandman et al.,    Human phosphatase inhibitor protein.-   U.S. Pat. No. 6,579,994, issued Jun. 17, 2003, Sankarasubbier N et    al., Process for preparation of 2-methyl-1,4-naphthoquinone.-   U.S. Patent Application Publication No. US 2003/0170187, published    Sep. 11, 2003, Marchal A, Skin treatments containing nano-sized    vitamin K.-   U.S. Patent Application Publication No. US 2004/0138218 A1,    published Jul. 15, 2004, Pallen et al., Novel protein tyrosine    phosphatase inhibitor.

1-27. (canceled)
 28. A pharmaceutical composition comprising an amountof a vitamin K3 effective to treat or prevent a skin rash secondary toan anti-epidermal growth factor receptor (EGFR) therapy. 29-41.(canceled)
 42. A method for treating or preventing a skin or mucosalulceration in a subject, the method comprising applying to the skin ormucosal ulceration an amount of a vitamin K1 or vitamin K3 effective totreat the skin or mucosal ulceration.
 43. The method of claim 42,wherein the skin or mucosal ulceration is ulcerative colitis,chemotherapy-induced oral mucosistis, or radiotherapy-induced oralmucosistis. 44-45. (canceled)
 46. The method of claim 42, whereinvitamin K3 is applied to the skin or mucosal ulceration at aconcentration of at least 15 μg/ml.
 47. The method of claim 42, whereinvitamin K3 is applied to the skin or mucosal ulceration at aconcentration of 75 μg/ml to 100 μg/ml.
 48. The method of claim 42,wherein vitamin K1 is applied to the skin or mucosal ulceration at aconcentration of at least 450 μg/ml.
 49. A method for treating orpreventing a skin rash secondary to an anti-epidermal growth factorreceptor (EGFR) therapy in a subject receiving said therapy, the methodcomprising applying a phosphatase inhibitor to the skin rash in anamount effective to treat the skin rash. 50-65. (canceled)
 66. Apharmaceutical composition comprising an amount of a phosphataseinhibitor effective to treat or prevent a skin rash secondary to ananti-epidermal growth factor receptor (EGFR) therapy. 67-69. (canceled)70. A method for treating or preventing a skin or mucosal ulceration ina subject, the method comprising applying to the skin or mucosalulceration an amount of a phosphatase inhibitor effective to treat theskin or mucosal ulceration, wherein the phosphatase inhibitor inhibitsdephosphorylation of EGFR.
 71. (canceled)
 72. The method of claim 42,wherein the vitamin K1 or vitamin K3 is formulated in a pharmaceuticalformulation suitable for topical administration.